Exome sequencing identifies novel variants associated with non-syndromic hearing loss in the Iranian population

Autosomal recessive non-syndromic hearing loss (ARNSHL) is a public health concern in the Iranian population, with an incidence of 1 in 166 live births. In the present study, the whole exome sequencing (WES) method was applied to identify the mutation spectrum of NSHL patients negative for GJB2 gene mutations. First, using ARMS PCR followed by Sanger sequencing of the GJB2 gene, 63.15% of mutations in patients with NSHL were identified. Among the identified mutations in GJB2:p.Val43Met and p.Gly21Arg were novel. The remaining patients were subjected to WES, which identified novel mutations including MYO15A:p.Gly39LeufsTer188, ADGRV1:p.Ser5918ValfsTer23, MYO7A: c.5856+2T>c (splicing mutation), FGF3:p.Ser156Cys. The present study emphasized the application of WES as an effective method for molecular diagnosis of NSHL patients negative for GJB2 gene mutations in the Iranian population.


Introduction
Hearing loss (HL) is considered one of the most frequent sensory impairments in humans, affecting all age groups and genders with an incidence of 1-2 in 1000 neonates in the world [1]. It has been documented that approximately 50-70% of cases of HL are related to genetic causes [2]. Hereditary HL can be regarded as syndromic or non-syndromic. Non-syndromic hearing loss is a partial or total loss of hearing with no other signs and symptoms. In contrast, syndromic hearing loss occurs with signs and symptoms affecting other parts of the body. According to severity, HL is classified into four grades (mild, moderate, severe, and profound) [3]. Nonsyndromic hearing loss (NSHL) shows different modes of inheritance, including autosomal recessive (AR), autosomal dominant (AD), X-linked, and mitochondrial [4]. AR transmission accounts for 75%-85% of all cases, while AD inheritance accounts for 15%-25% of cases. A small proportion of cases (1%-2%) show X-linked or mitochondrial inheritance [5].
To date, over 60 mapped loci have been identified for NSHL as a highly heterogeneous condition. The reports indicate that mutations in one locus, DFNB1 (13q11-12) which contains GJB2 (NM_004004.5) and GJB6 (NM_001110219.2) genes, make up 50% of the etiology in many populations [6]. GJB2 encodes the connexin 26 protein (Cx26), which is a member of the connexin protein family, forms channels called gap junctions, and is involved in inner ear homeostasis through the recycling of potassium ions. The GJB6 gene structure is thought to be relatively simple, consisting of only two exons, with one untranslated exon (exon 1) [7]. Overexpression of GJB2 has been found to be associated with a poor prognosis in several human cancers [8][9][10]. It has been reported that GJB2 expression was negatively associated with progression status in breast cancer tissues and may function as a regulator of breast tumorigenesis. Moreover, the knockdown of GJB2 in human breast cancer cell lines using shRNA resulted in a significant decrease in the proliferative ability and an increase in the migratory ability of breast cancer cells [11]. Until now, more than 100 pathogenic variants in the GJB2 gene have been reported to cause ARNSHL [12]. The prevalence of GJB2 mutations is different between populations. For instance, in Caucasians, c.35delG is the most frequent mutation leading to deafness. Distribution of the c.35delG frequency in carriers in this population is as high as 2-4% [13]. However, c.167delT in the Ashkenazi Jewish [14], c.235delC in the Japanese [15] and p.Trp24* in the Indians [16] are the most common mutations. Several studies have shown that the GJB2 mutations have contributed to 16-18% of ARNSHL in the Iranian population [17][18][19] , respectively. However, in this population, the frequency of GJB2 mutation varies in different regions. In the South part of Iran, it is less than 4% but increases to 27.5% in the North and Northwest of Iran [26]. Moreover, studies on a large number of families with ARNSHL from Sistan and Baluchistan province in the Southeast of Iran indicated that 7% of the population had mutations in the GJB2 gene. Interestingly, the GJB2:c.35delG mutation which is the most frequent mutation in different regions of Iran was not observed in the Sistan and Baluchistan province and the most prevalent mutation was p. Trp24*(80% from all GJB2 reported mutations) [27]. These findings were comparable to the presented data from a Pakistani population that is geographically close to Sistan and Baluchistan province of Iran [24]. Additionally, about 26% of Iranian Azeri (northwest of Iran) ARNSHL patients' etiology is due to mutations in the GJB2 gene [28]. In this population, it has been stated that c.35delG accounted for about 62% of the GJB2 mutations. Moreover, the results of a study on a Turkish population which is close to the Iranian Azeri population, were in line with this study [23]. Together, these reports showed the presence of an unequal distribution in the prevalence of GJB2-related ARNSHL throughout the Iranian population.
However, there is no defined strategy for the diagnosis of NSHL disease-causing mutations in this population. In the present study, using ARMS-PCR followed by Sanger and whole exome sequencing, it was possible to detect almost the entire disease-causing mutations among the patients affected with NSHL in the Iranian population.

Sample collection
A total of 76 patients affected with non-syndromic hearing loss (NSHL) mostly from consanguineous marriage (90.79%) which were referred to Isfahan medical genetics center (www. isfahangenetics.com) were included in this study. All the patients were diagnosed with NSHL and referred by the audiologist. The clinical history of each proband was explored to ensure that the hearing loss was not a result of infection, trauma, acoustic trauma, or ototoxic drugs. The patients had no other clinical manifestation except hearing loss.
In this study, written consent was obtained from all participants and parental consent was obtained when the patient was a child. All procedures were approved by University of Isfahan institutional review board (IRB) for research and ethics approval (Approval Ref No. 790205). A total of 122 individuals was tested including 76 probands and 46 probands' family members who participated in the co-segregation analysis.
Blood samples were collected at Isfahan medical genetics center. Genomic DNA was extracted from peripheral blood mononuclear cells of patients and their family members by salting out procedure [29]. The DNA was quantified and stored at room temperature for daily experiments and kept at -20˚C for future applications.

Genotyping
Mutation analysis was performed first for the detection of GJB2: c.35delG mutation using amplification refractory mutation analysis system PCR (ARMS-PCR) [30]. Two PCR assays using the normal or mutant primer along with the common and control primer were used (Table 1). Then, Sanger sequencing of exon 2 of GJB2 was performed for samples that were negative for any pathogenic or likely pathogenic variants in ARMS-PCR. The primer used for ARMS-PCR and sequencing (Table 1) were designed using Primer3 (bioinfo.ut.ee/primer3-0.4.0/). PCR reactions for sequencing were performed under the following conditions: initial denaturation at 95˚C / 4 minutes, followed by 30 cycles of denaturation at 95˚C/30 seconds, annealing at 59˚C/30 seconds, elongation at 72˚C/45 seconds, and extension at 72˚C/5 minutes.
According to the American College of Medical Genetics and Genomics (ACMG) guidelines, different criteria like frequency of variations in the population database, mode of inheritance, and prediction software were used to filter out detected variants [

Results and discussion
This study was performed on 76 patients diagnosed with NSHL from the central provinces of Iran (Patients' demographic data mentioned at S1 Table). In the first step, all the patients were screened for GJB2:c.35delG mutation using the ARMS-PCR method. The results showed that 35 patients (46.05%) were homozygous for this mutation. The patients who were homozygous for 35delG were affected by profound hearing loss (consisting of 10 males and 25 females). Interestingly, except for one family, most positive cases for 35delG were from consanguineous marriages. Next, to find other mutations in GJB2, exon 2 of the GJB2 gene (the only coding exon of the gene) was fully sequenced in the remaining 41 negative cases. Among the patients examined, six patients had homozygous variants, and seven had only one heterozygous mutation for GJB2. As mentioned in Table 2, all variants identified in exon 2 of GJB2 were classified pathogenic or likely pathogenic based on ACMG guideline. However, for 28 patients no mutation was detected. All the mutations identified using Sanger sequencing were presented in Table 2. The results from screening using Sanger sequencing of GJB2: exon2 showed that 13 of 76 cases (17.10%), were positive. Among these positive cases, 6 cases were homozygous and 7 were heterozygous. It is important to note that all the cases except 28867 and 222597 were from the results of consanguineous parents. Interestingly, in patient 28867, the identified mutation (rs28931593) is a pathogenic variant that was reported before and causes an autosomal dominant phenotype. However, in case 222597, the identified mutation (rs529500747) is a variant that its association with NSHL is controversial [40][41][42] (Table 3). This indicates that it is likely that there be a second mutation (variation) in other related genes such as GJB3 or GJB6 that may be the cause of the deafness in a compound heterozygous situation.
To identify disease-causing mutations in the remaining negative cases, whole exome sequencing (WES) was carried out. Among the patients analyzed by WES, one pathogenic or likely pathogenic variant related to hearing loss was identified for 85.7% of the patients. However, 4 patients (14.28%) were negative for any point mutation and/or small deletions. These results were presented in Table 4. Allele frequency, scores of pathogenicity prediction software related to identified variants by WES mentioned at S2 Table. Of these mutations which were identified by WES, four mutations were not previously reported and could be considered novel (Fig 1). Segregation analysis of novel variants in the proband's available family members is mentioned in Fig 1. These mutations consisted of i) MYO15A:c.115_116del and MYO15A:c.118_119del; and ii) ADGRV1:c.17752_17755del which were both deletion mutations with stop gain codon, iii) MYO7A:c.5856+2T>c which could result in the disruption of splicing and iv) FGF3:c.467C>G which is a missense mutation. An alternative variant at this position of FGF3 (chr11:69625327 A)G (Ser156Pro)) was previously reported and classified as pathogenic by ClinVar [47]. OTOF:c.1981dup is a duplication variant that causes a stop gain codon. It has been previously reported in an Iranian family, but yet has not been included in any databases [45].
The clinical significance of the variants identified by WES was included in Table 4. As presented, of 24 cases that were positive by WES, 19 patients had pathogenic variants (79.16%), 3 cases (12.5%) were likely pathogenic and 2 cases (8.3%) had a variation with uncertain significance classification (see Table 5).
Mutations in myosin-VIIa (MYO7A) cause three types of diseases: I) Deafness, autosomal dominant 11 (601317), II) Deafness, autosomal recessive 2 (600060) III) Usher syndrome, type 1B (276900). In this study, all three types have been observed: Proband 263097 is a male with an onset of mild hearing loss at 4 years old, now he is 13 years old. WES identified a heterozygous substitution (Pro1220Leu) in MYO7A that was reported as a variant of uncertain significance in ClinVar in association with hearing loss but based on ACMG guideline this variant was classified as likely pathogenic. Proband 261505 is 10 years old boy with an onset of profound sensorineural hearing at birth and retinitis pigmentosa at 6 years old. A pathogenic homozygous variant (Gly25Arg) at MYO7A was identified for him. Proband 250906 is 55 years old male with profound hearing loss from birth. WES identified a novel variant in MYO7A (c. 5856+2 T>C) that may disrupt the splicing of Exon 42 which has a severe effect on protein domains.  The majority of MYO15A variants are associated with a congenital severe to profound nonsyndromic hearing loss phenotype except for some exon 2 variants [48]. In this study, four variants in MYO15A have been identified: three mutations are termination mutation (exon 2, 4,0, and 66) and one substitution (exon 9). All four homozygous mutations identified in this study are classified as pathogenic or likely pathogenic based on ACMG guideline and all patients are affected by congenital severe to profound non-syndromic hearing loss (Table 4) OTOF-related deafness is characterized by two phenotypes: prelingual nonsyndromic auditory neuropathy spectrum disorder (ANSD) and, less frequently, temperature-sensitive auditory neuropathy spectrum disorder (TS-ANSD). Three pathogenic variants were identified in OTOF consisting of one deletion, one duplication, and one substitution. c.1966del and c.3515G>A are pathogenic variants that were reported previously, but c.1981dup is a novel Abbreviations and symbols are as follows: The arrow represents the proband, black represents deafness, the cross represents death, the question mark represents the unknown and the dot represents carrier. For details regarding each variant, see Table 3. https://doi.org/10.1371/journal.pone.0289247.g001 variant that causes termination of the protein. All three patients are affected with congenital severe and profound SNHL. Moreover, among the variations identified COL11A2:NM_080680:exon8:c.966dupC:p. Thr323HisfsTer19 is a variant with conflicting interpretations of pathogenicity, which was identified in two patients from two different families with consanguineous marriages (Table 2). Interestingly, this variant was reported as a cause of hearing loss in Ellis-van Creveld Syndrome with hearing loss in an Iranian child. In this patient with two distinct phenotypes, a mutation in EVC2 (c.2653C>T; p.Arg885*) was the reason for Ellis-van Creveld Syndrome and COL11A2:c.966dupC for hearing loss [49]. Therefore, this study along with two more cases and co-segregation in their families can support the pathogenicity of this variant (Fig 2).
Previous studies have shown the association of mutations in GJB2, SLC26A4, and TECTA genes with high priority and in genes MYO15A, ILDR1, TMC1, PJVK, LRTOMT, MYO7A, OTOF, and MARVELD2 with less significance with ARNSHL in the Iranian population [26]. The data from the present study indicated that GJB2 (c.35delG: 46.05% of cases), MYO15A (4 families), OTOF (3 families) and MYO7A (3 families) could be considered as more distinct pathogenic variants in the Iranian population (Table 2). Mutations in GJB2, especially GJB2: c.35delG, were of most importance in the central regions of Iran and its frequency is in line with north of Iran but not Southeast of Iran. The variants reported in the present study along with other frequent variants will help genetic counseling and family planning for families with deaf patients, which is expected to affect the frequency of deaf individuals in the Iranian population.
All the Iranian patients included in the present study were non-syndromic and had no history of cancer. Moreover, other studies in Iran did not report any cancer associated with GJB2related ARNSHL [50]. This might be explained by the notion that the increased expression of GJB2 has been associated with the progression of different cancers, indicating an oncogenic role for the GJB2 protein [11]. However, in GJB2-related ARNSHL patients, usually there is a lack of normal expression (abnormal mRNA and or protein expression) of GJB2 protein.
Besides, it is speculated that defects in the GJB2 gene might function toward the prevention of cancer in GJB2-related ARNSHL which needs further investigation.
According to the observation that even non-consanguineous parents were carriers of mutations, it is assumed that the funder effects might be considered one of the important factors in the prevalence of the ARNSHL disease in the Iranian population. Moreover, the study population which is almost from Isfahan province encompasses various sects today. The majority of the people in the province are Persian but Bakhtiari Lurs, Kurds, Georgians, Armenians, Qashqais, and Persian Jews. This may indicate that the population is almost isolated and the inbreeding rate within each ethnicity is high. Therefore, the effects of possible genetic drift could be considered. It seems that among the world's most heterogeneous populations, Iran has received a great deal of attention as a potential risk factor for different autosomal recessive disorders.

Conclusion
In the present study, for the first time, using whole exome sequencing (WES), the mutation spectrum of patients with NSHL who were negative for mutations in the GJB2 gene was depicted and novel mutations were identified in the Iranian population. Despite the high allele frequency of GJB2:35delG in the central region of Iran, the data showed that WES could be considered a convenient and cost-effective tool for the identification of the genetic cause of heterogenic diseases like NSHL compare to ARMS-PCR and Sanger sequencing.
Supporting information S1